Vectors for tissue-specific replication

ABSTRACT

The invention generally relates to targeted gene therapy using recombinant vectors and particularly adenovirus vectors. The invention specifically relates to replication-conditional vectors and methods for using them. Such vectors are able to selectively replicate in a target tissue to provide a therapeutic benefit from the presence of the vector per se or from heterologous gene products expressed from the vector and distributed throughout the tissue. In such vectors, a gene essential for replication is placed under the control of a heterologous tissue-specific transcriptional regulatory sequence. Thus, replication is conditioned on the presence of a factor(s) that induces transcription or the absence of a factor(s) that inhibits trancription of the gene by means of the transcriptional regulatory sequence with this vector; therefore, a target tissue can be selectively treated.

This application is a continuation of U.S. application Ser. No.09/210,936, filed Dec. 15, 1998, which is a continuation of U.S.application Ser. No. 08/849,117, filed Aug. 1, 1997, now U.S. Pat. No.5,998,205, which is the U.S. national phase under 35. U.S.C. § 371 ofPCT application PCT/US95/15455, filed Nov. 28, 1995, which is acontinuation-in-part of U.S. application Ser. No. 08/487,992, filed Jun.7, 1995, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 08/348,258, filed Nov. 28, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to targeted gene therapy usingrecombinant vectors and particularly adenovirus vectors. The inventionspecifically relates to replication-conditional vectors and methods forusing them. Such vectors are able to selectively replicate in a targettissue to provide a therapeutic benefit from the presence of the vectorper se or from heterologous gene products expressed from the vector anddistributed throughout the tissue. In such vectors, a gene essential forreplication is placed under the control of a heterologoustissue-specific transcriptional regulatory sequence. Thus, replicationis conditioned on the presence of a factor(s) that induces transcriptionor the absence of a factor(s) that inhibits transcription of the gene bymeans of the transcriptional regulatory sequence. With this vector,therefore, a target tissue can be selectively treated. The inventionalso relates to methods of using the vectors to screen a tissue for thepresence or absence of transcriptional regulatory functions that permitvector replication by means of the transcriptional regulatory sequence.The invention also relates to cells for producing recombinantreplication-conditional vectors useful for targeted gene therapy.

2. Background Art

Targeting Vectors

One of the major goals for therapeutic use of exogenous genes has beencell targeting with high specificity. General approaches have includedsystemic introduction of DNA, DNA-protein complexes, and liposomes. Insitu administration of retroviruses has also been used for cells thatare actively replicating.

However, because of the lack of, or significantly low, cell-specificityand inefficient gene transfer, the targeting of desired genes tospecific cells in an organism has been a major obstacle for exogenousgene-based therapy. Thus, the use of such genes has been limited.

Tumor cells are among those cell types for which it would be especiallydesirable to provide a means for exogenous gene targeting. In anembodiment of the present invention, compositions and methods areprovided to deliver exogenous genes to tumor cells safely andefficiently.

Adenoviruses Generally

Adenoviruses are nonenveloped, regular icosohedrons. The protein coat(capsid) is composed of 252 capsomeres of which 240 are hexons and 12are pentons. Most of the detailed structural studies of the adenoviruspolypeptides have been done for adenovirus types 2 and 5. The viral DNAis 23.85×10⁶ daltons for adenovirus 2 and varies slightly in sizedepending on serotype. The DNA has inverted terminal repeats and thelength of these varies with the serotype.

The replicative cycle is divided into early (E) and late (L) phases. Thelate phase defines the onset of viral DNA replication. Adenovirusstructural proteins are generally synthesized during the late phase.Following adenovirus infection, host DNA and protein synthesis isinhibited in cells infected with most serotypes. The adenovirus lyticcycle with adenovirus 2 and adenovirus 5 is very efficient and resultsin approximately 10,000 virions per infected cell along with thesynthesis of excess viral protein and DNA that is not incorporated intothe virion. Early adenovirus, transcription is a complicated sequence ofinterrelated biochemical events, but it entails essentially thesynthesis of viral RNAs prior to the onset of viral DNA replication.

The organization of the adenovirus genome is similar in all of theadenovirus groups and specific functions are generally positioned atidentical locations for each serotype studied. Early cytoplasmicmessenger RNAs are complementary to four defined, noncontiguous regionson the viral DNA These regions are designated (E1-E4). The earlytranscripts have been classified into an array of immediate early (E1a),delayed early (E1b, E2a, E2b, E3 and E4), and intermediate (IVa2.IX)regions.

The E1a region is involved in transcriptional transactivation of viraland cellular genes as well as transcriptional repression of othersequences. The E1a gene exerts an important control function on all ofthe other early adenovirus messenger RNAs. In normal tissues, in orderto transcribe regions E1b, E2a, E2b, E3, or E4 efficiently, active, E1aproduct is required. However, as discussed below, the E1a function maybe bypassed. Cells may be manipulated to provide E1a-like functions ormay naturally contain such functions. The virus may also be manipulatedto bypass the functions as described below.

The E1b region is, required for the normal progression of viral eventslate in infection. The E1b product acts in the host nucleus. Mutantsgenerated within the E1b sequences exhibit diminished late viral mRNAaccumulation as well as impairment in the inhibition of host cellulartransport normally observed late in adenovirus infection (Berkner, K.L., Biotechniques 6:616-629 (1988)). E1b is required for alteringfunctions of the host cell such that processing and transport areshifted in favor of viral late gene products. These products then resultin viral packaging and release of virions. E1b produces a 19 kD proteinthat prevents apoptosis. E1b also produces a 55 kD protein that binds top53.

For a complete review on adenoviruses and their replication, seeHorwitz, M. S., Virology 2d ed., Fields, B. N., eds., Raven PressLimited, New York (1990), Chapter 60, pp. 1679-1721.

Adenovirus as Recombinant Delivery Vehicle

Adenovirus provides advantages as a vector for adequate gene deliveryfor the following reasons. It is a double stranded DNA nonenvelopedvirus with tropism for the human respiratory system and gastrointestinaltract. It causes a mild flu-like disease. Adenoviral vectors enter cellsby receptor mediated endocytosis. The large (36 kilobase) genome allowsfor the removal of genes essential for replication and nonessentialregions so that foreign DNA may be inserted and expressed from the viralgenome. Adenoviruses infect a wide variety of cell types in vivo and invitro. Adenoviruses have been used as vectors for gene therapy and forexpression of heterologous genes. The expression of viral or foreigngenes from the adenovirus genome does not require a replicating cell.Adenovirus vectors rarely integrate into the host chromosome; theadenovirus genome remains as an extrachromosomal element in the cellularnucleus. There is no association of human malignancy with adenovirusinfection; attenuated strains have been developed and have been used inhumans for live vaccines.

For a more detailed discussion of the use of adenovirus vectors for genetherapy, see Berkner, K. L., Biotechniques 6:616-629 (1988); Trapnell,B. C., Advanced Drug Delivery Reviews 12:185-199. (1993).

Adenovirus vectors are generally deleted in the E1 region of the virus.The E1 region may then be substituted with the DNA sequences ofinterest. It was pointed out in a recent article on human gene therapy,however, that “the main disadvantage in the use of adenovirus as a genetransfer vector is that the viral vector generally remains episomal anddoes not replicate, thus, cell division leads to the eventual loss ofthe vector from the daughter cells” (Morgan, R. A., et al., AnnualReview of Biochemistry 62:191-217. (1993)) (emphasis added).

Non-replication of the vector leads not only to eventual loss of thevector without expression in most or all of the target cells but alsoleads to insufficient expression in the cells that do take up thevector, because copies of the gene whose expression is desired areinsufficient for maximum effect. The insufficiency of gene expression isa general limitation of all non-replicating delivery vectors. Thus, itis desirable to introduce a vector that can provide multiple copies of agene and hence greater amounts of the product of that gene. The presentinvention overcomes the disadvantages discussed above by providing atissue-specific, and especially a tumor-specific replicating vector,multiple DNA copies, and thus increased amounts of gene product.

Production of Adenoviral Vectors

Adenoviral vectors for recombinant gene expression have been produced inthe human embryonic kidney cell line 293 (Graham, F. L. et al., J. Gen.Virol. 36:59-72 (1977)). This cell line, initially transformed withhuman adenovirus 5, now contains the left end of the adenovirus 5 genomeand expresses E1. Therefore, these cells are permissive for growth ofadenovirus 2 and adenovirus 5 mutants defective in E1 functions. Theyhave been extensively used for the isolation and propagation of E1mutants. Therefore, 293. cells have been used for helper-independentcloning and expression of adenovirus vectors in mammalian cells. E1genes integrated in cellular DNA of 293 cells are expressed at levelswhich permit deletion of these genes from the viral vector genome. Thedeletion provides a nonessential region into which DNA may be inserted.For a review, see, Young, C. S. H., et al. in The Adenoviruses,Ginsberg, H. S., ed., Plenum Press, New York and London (1984), pp.125-172.

However, 293 cells are subject to severe limitations as producer cellsfor adenovirus vectors. Growth rates are low. Titres are limited,especially when the vector produces a heterologous gene product thatproves toxic for the cells. Recombination with the viral-E1 sequence inthe genome can lead to the contamination of the recombinant defectivevirus with unsafe wild-type virus. The quality of certain viralpreparations is poor with regard to the ratio of virus particle toplaque forming unit. Further, the cell line does not support growth ofmore highly deleted mutants because the expression of E1 in combinationwith other viral genes in the cellular genome (required to complementthe further deletion), such as E4, is toxic to the cells. Therefore, theamount of heterologous, DNA that can be inserted into the viral genomeis limited in these cells. It is desirable, therefore, to produceadenovirus vectors for gene therapy in a cell that cannot producewild-type recombinants and can produce high titres of high-qualityvirus. The present invention overcomes these limitations.

SUMMARY OF THE INVENTION

In view of the limitations discussed above, a general object of theinvention is to provide novel vectors for tissue-specific vectorreplication and gene expression from the replicating vector.Accordingly, the invention is directed to a vector that contains a genewhich is essential for replication, and which gene is operably linked toa heterologous transcriptional regulatory sequence, such that a vectoris created whose replication is conditioned upon the presence of atrans-acting transcriptional regulatory factor(s) that permitstranscription from the transcriptional regulatory sequence, or theabsence of a transcriptional regulatory factor(s) that normally preventstranscription from that transcriptional regulatory sequence. Thus, theseregulatory sequences are specifically activated or derepressed in thetarget tissue so that replication of the vector proceeds in that tissue.

Another object of the invention is to provide tissue-specific treatmentof an abnormal tissue. Thus, a further object of the invention is toprovide a method to selectively distribute a vector in vivo in a targettissue, such that a greater number of cells are treated than would betreated with a non-replicating vector, and treatment is avoided orsignificantly reduced in non-target tissue. Accordingly, a method isprovided for selectively distributing a vector in a target tissue byintroducing the replication-conditional vector of the present inventioninto a target tissue that contains a transcriptional regulatoryfactor(s) that allows replication of the vector or is deficient in atranscription-inhibiting factor(s) that prevents replication of thevector.

For providing tissue-specific treatment, another object of the inventionis to selectively distribute a polynucleotide in a target tissue invivo. Accordingly, the invention is, directed to a method forselectively distributing a polynucleotide in a target tissue in vivo byintroducing the replication-conditional vector of the present invention,containing the polynucleotide, into the target tissue that contains atranscriptional regulatory factor(s) that allows replication of thevector or is deficient in a transcription-inhibiting factor(s) thatprevents replication of the vector.

For providing tissue-specific treatment, a further object of theinvention is to selectively distribute a heterologous gene product in atarget tissue. Accordingly, the replication-conditional vectors of thepresent invention are constructed so that they contain a heterologousDNA sequence encoding a gene product that is expressed in the vector.When the vector replicates in the target tissue, effective quantities ofthe desired gene product are also produced in the target tissue.

Another object of the invention is to provide a method to identifyabnormal tissue that can be treated by the vectors of the presentinvention. Therefore, a further object of the invention is to identify atissue in which the replication-conditional vectors of the presentinvention can be replicated by means of the transcriptional regulatorysequence contained on the vector. Accordingly, the invention is furtherdirected to a method wherein the replication-conditional vectors of thepresent invention are exposed to a given abnormal tissue. If that tissuecontains a transcriptional regulatory factor(s) that allows replicationof the vector or is deficient in a transcription-inhibiting factor(s)that prevents replication of the vector, then replication of the vectorwill occur and can be detected. Following identification of such atissue, targeted treatment of that tissue can be effected bytissue-specific transcription and the consequent vector replication inthat tissue in vivo.

Thus, a method is provided for assaying vector utility for tissuetreatment comprising the steps of removing a tissue biopsy from apatient, explanting the biopsy into tissue culture, introducing areplication-conditional vector into the cells of the biopsy, andassaying for vector replication in the cells.

Another object of the invention, is to provide producer cell lines forvector production. Preferably, the cell lines have one or more of thefollowing characteristics: high titer virus production, resistance totoxic effects due to heterologous gene products expressed in the vector,lack of production of wild-type virus contaminating the viruspreparation and resulting from recombination between integrated viralsequences and vector sequences, growth to high density and insuspension, unlimited passage potential, high growth rate, and bypermitting the growth of highly deleted viruses that are impaired forviral functions and able to accommodate large pieces of heterologousDNA.

Accordingly, in a further embodiment of the invention, a cell line isprovided containing the replication-conditional vector of the presentinvention, the cells of which cell line contain a transcriptionalregulatory factor(s) that allows replication of the vector or isdeficient in a transcription-inhibiting factor(s) that preventsreplication of the vector.

In further embodiments of the invention, the cell line contains nucleicacid copies of the replicated vector. In other embodiments, the cellline contains virions produced in the cell by replication in the cell ofthe replication-conditional vector.

In further embodiments, a method is provided for producing areplication-conditional vector or virion comprising the steps ofculturing the producer cell line described above and recovering thevector or virion from the cells. In still further embodiments, a methodis provided for producing replication-conditional virions free ofwild-type virions or viral vectors free of wild-type vectors, comprisingthe steps of culturing the producer cell line described above andrecovering the replication-deficient virions or vectors from the cells.

In a preferred methods of treatment and diagnosis, the tissue isabnormally proliferating, and especially is tumor tissue. However, themethods are also directed to other abnormal tissue as described herein.

In preferred embodiments of the invention, the replication-conditionalvector is a DNA tumor viral vector. In a further preferred embodiment,the DNA tumor viral vector is a vector selected from the groupconsisting of herpesvirus, papovavirus, papillomavirus, parvovirus, andhepatitis virus vectors. In a most preferred embodiment, the vector isan adenovirus vector. However, it is to be understood that potentiallyany vector source, is useful if it contains a gene essential forreplication that can be operably linked to a tissue-specifictranscriptional regulatory sequence.

In further methods of treatment and diagnosis, the vector is introducedinto the tissue by infection.

Replication can be vector nucleic acid replication alone or can alsoinclude virus replication (i.e., virion production). Thus, either DNA orvirions or both may be distributed in the target tissue.

In a further preferred embodiment of the invention, a gene in theadenovirus E1 region is operably linked to the tissue-specifictranscriptional regulatory sequence. Preferably, the E1a or E1b gene isoperably linked to the tissue-specific transcriptional regulatorysequence.

In a further embodiment of the invention, the vector encodes aheterologous gene product. This heterologous gene product is expressedfrom the vector replicating in the target tissue.

In a further embodiment of the methods of treatment, the heterologousgene product is toxic for the target tissue.

In a further embodiment of the methods, the heterologous gene productacts on a non-toxic prodrug, converting the non-toxic prodrug into aform that is toxic for the target tissue. Preferably, the toxin hasanti-tumor activity or eliminates cell proliferation.

In preferred embodiments of the invention, the transcriptionalregulatory sequence is a promoter. Preferred promoters include, but arenot limited to, carcinoembryonic antigen (CEA), DE3, α-fetoprotein(AFP), Erb-B2, surfactant, and especially lung surfactant, and thetyrosinase promoter. However, any genetic control region that controlstranscription of the essential gene can be used to activate (orderepress) the gene. Thus, other genetic control elements, such asenhancers, repressible sequences, and silencers, can be used to regulatereplication of the vector in the target cell. The only requirement isthat the genetic element be activated, derepressed, enhanced, orotherwise genetically regulated by factors in the host cell and, withrespect to methods of treatment, not in the non-target cell. Preferredenhancers include the DF3 breast cancer-specific enhancer and enhancersfrom viruses and the steroid receptor family. Other preferredtranscriptional regulatory sequences include NF1, SP1, AP1, and FOS/JUN.

In further embodiments, promoters are not necessarily activated byfactors in the target tissue, but are derepressed by factors present inthe target tissue. Thus, in the target tissue, repression is lifted.

Transcriptional regulatory factors include, but are not limited to,transactivating factors produced by endogenous viral sequences such asfrom cytomegalovirus (CMV), HIV, Epstein-Barr virus (EBV), Herpessimplex virus (HSV), SV40, and other such viruses that are pathogenic inmammals and, particularly, in humans.

Methods for making such vectors are well known to the person of ordinaryskill in the art. The art adequately teaches the construction ofrecombinant vectors with deletions, or modifications in specific codingsequences and operable linkage to a heterologous transcription controlsequence such that expression of a desired coding region is undercontrol of the heterologous transcriptional regulatory sequence. Manyviral sequences have been adequately mapped such that it is routine toidentify a gene of choice and use appropriate well known techniques(such as homologous recombination of the virus with deleted orotherwise, modified plasmids) to construct the vectors fortissue-specific replication and expression.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B. Cloning of pAVE1a02i: pAVSAFP.TK1 was digested withNheI/MunI. A 10667 bp fragment was isolated. pSE280-E1 was digested withSpeI/MunI. A 3397 bp fragment was isolated. The isolated fragments wereligated to form pAVE1a02i.

FIG. 2A-C. PCR identification of recombinant adenovirus with E1aexpressed from the hepatoma-specific AFP promoter. FIG. 2A shows thatviral plaques are produced by viral genomes containing the AFP promoteroperably linked to E1a. FIG. 2B shows that there was no contaminationwith wild-type virus. FIG. 2C shows that there was no contamination withAV1lacZ DNA.

FIG. 3A-F. Tissue specific adenovirus with E1a expressed from the AFPpromoter. The experiment shows cytopathic effects and spreading of celldeath following infection with the virus AVAFPE1a. FIGS. 3A-3C showuninfected controls in A549.30, A549, and HuH 7 cells, respectively.FIGS. 3D-3F show the results of infection with the virus in A549.30,A549, and HuH 7 cells, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

The term “abnormally proliferating” is intended to mean a cell having ahigher mitotic index than its normally-functioning counterpart, suchthat there is an abnormal accumulation of such cells.

The term “anti-tumor activity” is intended to mean any activity whichinhibits, prevents, or destroys the growth of a tumor.

The term “distributing” is intended to mean the spreading of a vectorand its attendant heterologous gene (product) (when present on thevector) throughout a target tissue, and especially throughout abnormallyproliferating tissue (non-malignant or malignant). The object of thedistribution is to deliver the vector, gene product or the effects ofthe gene product (as by a bystander effect, for example) tosubstantially all or a significant number of cells of the target tissue,so as to treat substantially the entire target tissue.

The term “enhancer” is used according to its art-recognized meaning. Itis intended to mean a sequence found in eukaryotes and certaineukaryotic viruses which can increase transcription from a gene whenlocated (in either orientation) up to several kilobases from the genebeing studied. These sequences usually act as enhancers when on the 5′side (upstream) of the gene in question. However, some enhancers areactive when placed on the 3′ side (downstream) of the gene. In somecases, enhancer elements can activate transcription from a gene with no(known) promoter.

The term “functional inactivation” is intended to mean a genetic lesionthat prevents the normal activity of a gene product. Thus, functionalinactivation could result from a mutation in the gene encoding the geneproduct. Such a lesion includes insertions, deletions, and base changes.Alternatively, functional inactivation may occur by the abnormalinteraction of the normal gene product with one or more other cellulargene products which bind to or otherwise prevent the functional activityof said gene product. Thus, the gene product may be a protein producedfrom a normal gene but which cannot perform its ordinary and normalfunction because of an interaction with a second factor.

The term “gene essential for replication” refers to a genetic sequencewhose transcription is required for the vector to replicate in thetarget cell.

The term “gene product” is intended to mean DNA, RNA, protein, peptides,or viral particles. Thus, the distribution, for the purposes of theinvention, is of any of these components.

The term “heterologous” means a DNA sequence not found in the nativevector genome. With respect to a “heterologous transcriptionalregulatory sequence”, “heterologous” indicates that the transcriptionalregulatory sequence is not naturally ligated to the DNA sequence for thegene essential for replication of the vector.

The term “promoter” is used according to its art-recognized meaning. Itis intended to mean the DNA region, usually upstream to the codingsequence of a gene or operon, which binds RNA polymerase and directs theenzyme to the correct transcriptional start site.

The term “replication” means, duplication of a vector. This duplication,in the case of viruses, can occur at the level of nucleic acid, or atthe level of infectious viral particle. In the case of DNA viruses,replication at the nucleic acid level is DNA replication. In the case ofRNA viruses, nucleic acid replication is replication into plus or minusstrand (or both). In the case if retroviruses, replication at thenucleic acid level includes the production of cDNA as well as thefurther production of RNA viral genomes. The essential feature isnucleic acid copies of the original viral vector. However, replicationalso includes the formation of infectious DNA or RNA viral particles.Such particles may successively infect cells in a given target tissuethus distributing the vector through all or a significant portion of thetarget tissue.

The term “replication-conditional vector” refers to a vector which whenintroduced into a tissue will not replicate unless a transcriptionalregulatory sequence in that vector is activated or derepressed in thattissue. That is, replication depends upon transcription by means of thattranscriptional regulatory sequence. Such a vector isreplication-conditional as described because it has been modified in thefollowing manner. A gene that is essential for replication has beenmodified by replacing the transcriptional regulatory sequence on whichtranscription of that gene normally depends with a heterologoustranscriptional regulatory sequence. This transcriptional to regulatorysequence depends upon the presence of transcriptional regulatory factorsor the absence of transcriptional regulatory inhibitors. The presence ofthese factors in a given tissue or the absence of such inhibitors in agiven tissue provides the replication-conditionality. Accordingly, thenative transcriptional regulatory sequence may be replaced with theheterologous transcriptional regulatory sequence. Alternatively, thenative transcriptional regulatory sequence may be disabled or rendereddysfunctional by partial removal (deletion) or other mutation (one ormore base changes, insertions, inversions, etc.).

The gene sequence may be a coding sequence. It may contain one or moreopen reading frames, as well as intron sequences. However, such asequence is not limited to a coding sequence, but includes sequencesthat are transcribed into RNA, which RNA is itself essential for vectorreplication. The essential feature is that the transcription of the genesequences does not depend on the native transcriptional regulatorysequences.

The term “silencer,” used in its art-recognized sense, means a sequencefound in eucaryotic viruses and eucaryotes which can decrease or silencetranscription of a gene when located within several kilobases of thatgene.

The term “tissue-specific” is intended to mean that the transcriptionalregulatory sequence to which the gene essential for replication isoperably linked functions specifically in that tissue so thatreplication proceeds in that tissue. This can occur by the presence inthat tissue, and not in non-target tissues, of positive transcriptionfactors that activate the transcriptional regulatory sequence. It canalso occur by the absence of transcription inhibiting factors thatnormally occur in non-target tissues, and prevent transcription as aresult of the transcription regulatory sequence. Thus, whentranscription occurs, it proceeds into the gene essential forreplication such that in that target tissue, replication of the vectorand its attendant functions occur.

As described herein, tissue specificity is particularly relevant in thetreatment of the abnormal counterpart of a normal tissue. Suchcounterparts include, but are not limited to, liver tissue and livercancer, breast tissue and breast cancer, melanoma and normal skintissue. Tissue specificity also includes the presence of an abnormaltissue type interspersed with normal tissue of a different tissue type,as for example in the case of metastases of colon cancer, breast cancer,and the like, into tissue such as liver. In this case, the target tissueis the abnormal tissue, and tissue specificity reflects the restrictionof vector replication to the abnormal tissue interspersed in the normaltissue. It is also to be understood that tissue specificity, in thecontext of treatment, is particularly relevant in vivo. However, asdescribed herein, ex vivo treatment and tissue replacement also fallswithin the concept of tissue specificity according to the presentinvention.

The term “transcriptional regulatory function” or “transcriptionalregulatory factor” is intended to mean any cellular function whosepresence activates the heterologous transcriptional regulatory sequencedescribed herein or whose absence permits transcription as a result ofthe transcriptional regulatory sequences described herein. It isunderstood that in the given target tissue, a tissue that “lacks thetranscriptional regulatory factor” or is “deficient” in thetranscriptional regulatory factor could refer to either the absence ofthe factor or the functional inactivation of the factor in the targettissue.

The term “transcriptional regulatory sequence” is used according to itsart-recognized meaning. It is intended to mean any DNA sequence whichcan, by virtue of its sequence, cause the linked gene to be either up-or down-regulated in a particular cell. In one embodiment of the presentinvention, the native transcriptional regulatory sequence is completelydeleted from the vector and replaced with a heterologous transcriptionalregulatory sequence. The transcriptional regulatory sequence may beadjacent to the coding region for the gene that is essential forreplication, or may be removed from it. Accordingly, in the case of apromoter, the promoter will generally be adjacent to the coding region.In the case of an enhancer, however, an enhancer can be found at somedistance from the coding region such that there is an intervening DNAsequence between the enhancer and the coding region. In some cases, thenative transcriptional regulatory sequence remains on the vector but isnon-functional with respect to transcription of the gene essential forreplication.

Various combinations of transcriptional regulatory sequences can beincluded in a vector. One or more may be heterologous. Further, one ormore may have the tissue-specificity. For example, a singletranscriptional regulatory sequence could be used to drive replicationby more than one gene essential for replication. This is the case, forexample, when the gene product of one of the genes drives transcriptionof the further gene(s). An example is a heterologous promoter linked toa cassette containing an E1a coding sequence (E1a promoter deleted) andthe entire E1b gene. In such a cascade, only one heterologoustranscriptional regulatory sequence may be necessary. When genes areindividually (separately) controlled, however, more than onetranscriptional regulatory sequence can be used if more than one suchgene is desired to control replication.

The vectors of the present invention, therefore, also includetranscriptional regulatory sequence combinations wherein there is morethan one heterologous transcriptional regulatory sequence, but whereinone or more of these is not tissue-specific. For example, onetranscriptional regulatory sequence can be a basal level constitutivetranscriptional regulatory sequence. For example, a tissue-specificenhancer can be combined with a basal level constitutive promoter.

Vectors

The preferred vectors of the present invention are adenoviral vectors.In a preferred embodiment of the invention, an adenovirus vectorcontains a tissue-specific transcriptional regulatory sequence linked toa gene in the E1 region.

In one embodiment, both E1a and E1b are operably linked to heterologoustissue-specific transcriptional regulatory sequences. In an alternativeembodiment, only E1a is linked; E1b remains intact. In still anotherembodiment, E1b is linked, and E1a remains intact or is deleted. In anycase, one or more tissue-specific and promoter-specific cellulartranscriptional regulatory factors allows virus replication to proceedby transcribing the E1a and/or E1b gene functionally linked to thepromoter. Further, either one or both of the E1b functions may be linkedto the transcriptional regulatory sequence.

In alternative embodiments, adenovirus vectors are provided with any ofthe other genes essential for replication, such as E2, E4, under controlof a heterologous transcriptional regulatory sequence.

The invention further embodies the use of plasmids and vectors havingonly the essential regions of adenovirus needed for replication witheither E1a, E1b 19 kDa gene, or E1b 55 kDa gene, or some combinationthereof, modified. Such a plasmid, lacking any structural genes, wouldbe able to undergo DNA replication. Accordingly, the vectors of theinvention may consist essentially of the transcriptional regulatorysequence and one or more genes essential for replication of the vector.In the case of viral vectors, the vectors may consist essentially of thetranscriptional regulatory sequence and the gene or genes essential forreplication or life-cycle functions of the virus. It is also understoodthat these vectors may also further consist essentially of a DNAsequence encoding one or more toxic heterologous gene products when suchvectors are intended as expression vectors for treatment.

In broader embodiments, the vector is derived from another DNA tumorvirus. Such viruses generally include, but are not limited to,Herpesviruses (such as Epstein-Barr virus, cytomegalovirus, Herpeszoster, and Herpes simplex), papillomaviruses, papovaviruses (such aspolyoma and SV40), and hepatitis viruses.

The alternative viruses preferably are selected from any group ofviruses in which the essential genes, for replication of the virus canbe placed under the control of a tissue-specific transcriptionalregulatory sequence. All serotypes are included. The only commonproperty of such viruses, therefore, is that they are transducible intotarget tissue, are genetically manipulatable, and are non-toxic when notreplicating.

The relevant viral gene(s) are those that are essential for replicationof the viral vector or of the virus. Examples of genes include, but arenot limited to, the E6 and E7 regions of human papilloma virus, 16 and18, T antigen of SV40, and CMV immediate early genes, polymerases fromretroviruses and the like. Essentially, these include any gene that isnecessary for the life cycle of the virus.

In further embodiments, the vector is derived from an RNA virus. Instill further embodiments, the vector is derived from a retrovirus. Itis understood, however, that potentially any replicating vector can bemade and used according to the essential design disclosed herein.

The vectors described herein can be constructed using standard molecularbiological techniques. Standard techniques for the construction of suchvectors are well-known to those of ordinary skill in the art, and can befound in references such as Sambrook et al., in Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y. (1989), or any of the myriadof laboratory manuals on recombinant DNA technology that are widelyavailable. A variety of strategies are available for ligating fragmentsof DNA, the choice of which depends on the nature of the termini of theDNA fragments and can be readily determined by the skilled artisan.

An adenovirus vector, in a preferred embodiment, is constructed first byconstructing, according to standard techniques, a shuttle plasmid whichcontains, beginning at the 5′ end, the “critical left end elements,”which include an adenoviral 5′ ITR, an adenoviral encapsidation signal,and an E1a enhancer sequence; a promoter (which may be an adenoviralpromoter or a foreign promoter); a tripartite leader sequence, amultiple cloning site (which may be as herein described); a poly Asignal; and a DNA segment which corresponds to a segment of theadenoviral genome. Such DNA segment serves as a substrate for homologousrecombination with a modified or mutated adenovirus. The plasmid mayalso include a selectable marker and an origin of replication. Theorigin of replication may be a bacterial origin of replication.Representative examples of such shuttle plasmids include pAVS6, asdiscussed herein and see Trapnell, B. et al., Adv. Drug Deliv. Rev12:185-189 (1994). A desired DNA sequence containing a heterologous genemay then be inserted into the multiple cloning site to produce a plasmidvector.

This construct then is used to produce an adenoviral vector. Homologousrecombination then is effected with a modified or mutated adenovirus inwhich one or more of the native transcriptional regulatory sequenceshave been deleted and replaced with the desired transcriptionalregulatory sequence. Such homologous recombination may be effectedthrough co-transfection of the plasmid vector and the modifiedadenovirus into a helper cell line by CaPO₄ precipitation.

Through such homologous recombination, a vector is formed which includesadenoviral DNA free of one or more of the native transcriptionalregulatory sequences. This vector may then be transfected into a helpercell line for viral replication and to generate infectious viralparticles. Transfections may take place by electroporation, calciumphosphate precipitation, microinjection, or through proteoliposomes.

The vector may include a multiple cloning site to facilitate theinsertion of DNA sequence(s) containing the heterologous gene into thecloning vector. In general, the multiple cloning site includes “rare”restriction enzyme sites; i.e., sites which are found in eukaryoticgenes at a frequency of from about one in every 10,000 to about one inevery 100,000 base pairs. An appropriate vector is thus formed bycutting the cloning vector by standard techniques at appropriaterestriction sites in the multiple cloning site, and then ligating theDNA sequence containing the heterologous gene into the cloning vector.

The DNA sequence encoding the heterologous gene product is under thecontrol of a suitable promoter. Suitable promoters which may be employedinclude, but are not limited to, adenoviral promoters, such as theadenoviral major late promoter; or heterologous promoters, such as thecytomegalovirus promoter, the Rous sarcoma virus promoter; induciblepromoters, such as the mouse mammary tumor virus (MMTV) promoter, themetallothionein promoter, heat shock promoters; the albumin promoter;the ApoE promoter; and the ApoAI promoter. It is to be understood,however, the scope of the present invention is not limited to specificforeign genes or promoters.

In one embodiment, the adenovirus may be constructed by using a yeastartificial chromosome containing an adenoviral genome according to themethod described in Ketner, et al., Proc. Nat. Acad. Sci. 91:6186-6190(1994), in conjunction with the teachings contained herein. In thisembodiment, the adenovirus yeast artificial chromosome is produced byhomologous recombination in vivo between adenoviral DNA and yeastartificial chromosome plasmid vectors carrying segments of theadenoviral left and right genomic termini. A DNA sequence containing theheterologous gene then may be cloned into the adenoviral DNA. Themodified adenoviral genome then is excised from the adenovirus yeastartificial chromosome in order to be used to generate infectiousadenoviral particles.

The infectious viral particles may then be administered in vivo to ahost. The host may be an animal host, including mammalian, non-humanprimate, and human hosts.

The viral particles may be administered in combination with apharmaceutically acceptable carrier suitable for administration to apatient The carrier may be a liquid carrier (for example, a salinesolution), or a solid carrier, such as, for example, microcarrier beads.

Treatment

In preferred embodiments, the methods are specifically directed to theintroduction into a target tissue of a replication-conditionaladenoviral vector. This vector selectively replicates in the cells ofthe target tissue. The replication is conditioned upon the function of atranscriptional regulatory sequence to which a viral gene is operablylinked, which gene is necessary for vector replication. Thus, in thetarget tissue, replication can occur because either a cellular functionin the target tissue allows transcription. Alternatively, there is adeficiency in a cellular function in the target tissue that normallyprevents or inhibits transcription. The presence or absence of suchfunctions provides the selectivity that allows the treatment of aspecific tissue with minimum effect on the surrounding tissue(s).

The present invention thus provides methods for selectively distributinga polynucleotide in a given tissue in vivo, significantly reducing oravoiding distribution in non-target tissue. The polynucleotide isprovided in the replication-conditional vector which is selectivelydistributed in the given tissue.

The present invention also provides methods for selectively expressing agene product in a given tissue, avoiding or significantly reducingexpression in non-target or non-tumor tissue. The invention providesmethods for distribution of the above-mentioned to a greater number oftarget cells than would be reached using a non-replicating vector.Successive infection provides a “domino effect” so that all orsubstantially all of the cells in the target tissue is reached. Cells inaddition to those first exposed to the polynucleotide, vector, or geneproduct, are thus potentially reached by the methods.

Such treatment is particularly necessary in cases in which surgicalintervention is not feasible. For example, in patients with abnormaltissue intimately associated with neural tissue, surgery may beprecluded or highly dangerous. Further, in the case of multiplemetastases or microscopic metastases, surgery is not feasible.

In the target tissue, DNA replication alone may occur. Late viralfunctions that result in packaging of vector DNA into virions may alsooccur.

The vector may be introduced into the target tissue as naked DNA or bymeans of encapsidation (as an infectious virus particle or virion). Inthe latter case, the distribution is accomplished by successiveinfections of cells in the tissue by the virus such that substantiallyall or a significant number of the daughter cells are infected.

Tissue specificity is particularly relevant with respect to targeting anabnormal counterpart of a particular tissue type while avoiding thenormal counterpart of the tissue, or avoiding surrounding tissue of adifferent type than the abnormal tissue, while treating the abnormaltissue. For example, the vectors of the present invention are useful fortreating metastases to the liver. One specific example is colon cancer,which often metastasizes into the liver. It has been found that evenwhen colon cancer metastasizes into the liver, the CEA promoter isactive in the cells of the metastases but not in normal liver cells.Accordingly, normal human adult liver should not support replication ofa virus that has viral genes essential for replication linked to thecolon cancer CEA-specific promoter. Replication should occur in theprimary cancer cells. Another example is breast cancer, which alsometastasizes to the liver. In this case, the DF3 mucin enhancer islinked to a gene essential for replication such as both E1a and E2aReplication should occur in breast cancer but not in normal liver. Afurther example is the α-fetoprotein promoter, which is active inhepatocellular carcinoma. This promoter is linked to a gene essentialfor replication. It has been found that the promoter is active only inthe hepatocellular carcinoma. Accordingly, a virus is used that has agene essential for replication linked to this promoter. Replicationshould be limited to hepatocellular carcinoma. A further example is thetyrosinase promoter. This promoter is linked to a gene essential forreplication. Replication should occur in melanoma and not in normalskin. In each case, replication is expected in the abnormal but not thenormal cells.

In a further embodiment of the invention, the vector encodes aheterologous gene product which is expressed from the vector in thetissue cells. The heterologous gene product can be toxic for the cellsin the targeted tissue or confer another desired property.

A gene product produced by the vector can be distributed throughout thetissue, because the vector itself is distributed throughout the tissue.Alternatively, although the expression of the gene product may belocalized, its effect may be more far-reaching because of a bystandereffect or the production of molecules which have long-range effects suchas chemokines. The gene product can be RNA, such as antisense RNA orribozyme, or protein. Examples of toxic products include, but are notlimited to, thymidine kinase in conjunction with ganciclovir.

A wide range of toxic effects is possible. Toxic effects can be director indirect. Indirect effects may result from the conversion of aprodrug into a directly toxic drug. For example, Herpes simplex virusthymidine kinase phosphorylates ganciclovir to produce the nucleotidetoxin ganciclovir phosphate. This compound functions as a chainterminator and DNA polymerase inhibitor, prevents DNA synthesis, andthus is cytotoxic. Another example is the use of cytosine deaminase toconvert 5′-fluorocytosine to the anti-cancer drug 5′-fluorouracil. For adiscussion of such “suicide” genes, see Blaese, R. M. et al., Eur. J.Cancer 30A:1190-1193 (1994).

Direct toxins include, but are not limited to, diphtheria toxin(Brietman et al., Mol. Cell. Biol. 10:474-479 (1990)), pseudomonastoxin, cytokines (Blankenstein, T., et al., J. Exp. Med. 173:1047-1052(1991), Colombo, M. P., et al., J. Exp. Med. 173:889-897 (1991), Leone,A., et al., Cell 65:25-35 (1991)), antisense RNAs and ribozymes (Zaia,J. A. et al., Ann. NY. Acad. Sci. 660:95-106 (1992)), tumor vaccinationgenes, and DNA encoding for ribozymes.

In accordance with the present invention, the agent which is capable ofproviding for the inhibition, prevention, or destruction of the growthof the target tissue or tumor cells upon expression of such agent can bea negative selective marker, i.e., a material which in combination witha chemotherapeutic or interaction agent inhibits, prevents or destroysthe growth of the target cells.

Thus, upon introduction to the cells of the negative selective marker,an interaction agent is administered to the host. The interaction agentinteracts with the negative selective marker to prevent, inhibit, ordestroy the growth of the target cells.

Negative selective markers which may be used include, but are notlimited to, thymidine kinase and cytosine deaminase. In one embodiment,the negative selective marker is a viral thymidine kinase selected fromthe group consisting of Herpes simplex virus thymidine kinase,cytomegalovirus thymidine kinase, and varicella-zoster virus thymidinekinase. When viral thymidine kinases are employed, the interaction orchemotherapeutic agent preferably is a nucleoside analogue, for example,one selected from the group consisting of ganciclovir, acyclovir, and1-2-deoxy-2-fluoro-β-D-arabinofuranosil-5-iodouracil (FIAU). Suchinteraction agents are utilized efficiently by the viral thymidinekinases as substrates, and such interaction agents thus are incorporatedlethally into the DNA of the tumor cells expressing the viral thymidinekinases, thereby resulting in the death of the target cells.

When cytosine deaminase is the negative selective marker, a preferredinteraction agent is 5-fluorocytosine. Cytosine deaminase converts5-fluorocytosine to 5-fluorouracil, which is highly cytotoxic. Thus, thetarget cells which express the cytosine deaminase gene convert the5-fluorocytosine to 5-fluorouracil and are killed.

The interaction agent is administered in an amount effective to inhibit,prevent, or destroy the growth of the target cells. For example, theinteraction agent is administered in an amount based on body weight andon overall toxicity to a patient. The interaction agent preferably isadministered systemically, such as, for example, by intravenousadministration, by parenteral administration, by intraperitonealadministration, or by intramuscular administration.

When the vectors of the present invention induce a negative selectivemarker and are administered to a tissue or tumor in vivo, a “bystandereffect” may result, i.e., cells which were not originally transducedwith the nucleic acid sequence encoding the negative selective markermay be killed upon administration of the interaction agent. Although thescope of the present invention is not intended to be limited by anytheoretical reasoning, the transduced cells may be producing adiffusible, form of the negative selective marker that either actsextracellularly upon the interaction agent, or is taken up by adjacent,non-target cells, which then become susceptible to the action of theinteraction agent. It also is possible that one or both of the negativeselective marker and the interaction agent are communicated betweentarget cells.

In one embodiment, the agent which provides for the inhibition,prevention, or destruction of the growth of the tumor cells is acytokine. In one embodiment, the cytokine is an interleukin. Othercytokines which may be employed include interferons andcolony-stimulating factors, such as GM-CSF. Interleukins include, butare not limited to, interleukin-1, interleukin-1β, andinterleukins-2-15. In one embodiment, the interleukin is interleukin-2.

In a preferred embodiment of the invention, the target tissue isabnormally proliferating, and preferably tumor tissue. The vector orvirus is distributed throughout the tissue or tumor mass.

All tumors are potentially amenable to treatment with the methods of theinvention. Tumor types include, but are not limited to hematopoietic,pancreatic, neurologic, hepatic, gastrointestinal tract, endocrine,biliary tract, sino-pulmonary, head and neck, soft tissue sarcoma andcarcinoma, dermatologic, reproductive tract, and the like. Preferredtumors for treatment are those with a high mitotic index relative tonormal tissue. Preferred tumors are solid tumors, and especially, tumorsof the brain, most preferably glioma.

The methods can also be used to target other abnormal cells, forexample, any cells in which are harmful or otherwise unwanted in vivo.Broad examples include cells causing autoimmune disease, restenosis, andscar tissue formation.

Further, treatment can be ex vivo. Ex vivo transduction of tumor cellswould overcome many of the problems with current viral delivery systems.Tissue is harvested under sterile conditions, dissociated mechanicallyand/or enzymatically and cultured under sterile conditions inappropriate media. Vector preparations demonstrated to be free ofendotoxins and bacterial contamination are used to transduce cells understerile conditions in vitro using standard protocols. The accessibilityof virus to cells in culture is currently superior to in vivo injectionand permits introduction of vector viral sequences into essentially allcells. Following removal of virus-containing media cells are immediatelyreturned to the patient or are maintained for several days in culturewhile testing for function or sterility is performed.

For example, patients with hypercholesterolemia have been treatedsuccessfully by removing portions of the liver, explanting thehepatocytes in culture, genetically modifying them by exposure toretrovirus, and re-infusing the corrected cells into the liver (Grossmanet al., 1994).

Viral transduction also has potential applications in the area ofexperimental medicine. Transient expression of biological modifiers ofimmune system function such as IL-2, IFN-γ, GM-CSF or the B7co-stimulatory protein has been proposed as a potential means ofinducing anti-tumor responses in cancer patients.

In broader embodiments, the vector is derived from another DNA tumorvirus. Such viruses generally include, but are not limited to,Herpesviruses (such as Epstein-Barr virus, cytomegalovirus, Herpeszoster, and Herpes simplex), papillomaviruses, papovaviruses (such aspolyoma and SV40), and hepatitis viruses.

The relevant viral gene(s) are those that are essential for replicationof the viral vector or of the virus. Examples of genes include, but arenot limited to, the E6 and E7 regions of human papilloma virus, 16 and18, T antigen of SV40, and CMV immediate early genes, polymerases fromretroviruses and the like. Essentially, these include any gene that isnecessary for the life cycle of the virus.

In further embodiments, the vector is derived from an RNA virus. Instill further embodiments, the vector is derived from a retrovirus. Itis understood, however, that potentially any replicating vector can bemade and used according to the essential design disclosed herein.

Diagnostic

It is important to know whether the vectors of the invention willreplicate in a specific tissue from a patient. If vector replication isfound to be beneficial for therapy, then a screen is provided for thosepatients who best respond to the therapy disclosed herein. If it isfound to be harmful, then there is a screen for prevention of thetreatment of patients who would have an adverse response to thetreatment. Currently, the only non-biological assays that are commonlyused are expression screening, PCR, and sequencing. These often resultin false, negatives, are time-consuming, expensive, and yield onlyinformation in the best of cases about the status of the genes and nottheir biological function.

Accordingly, a method is provided for identifying an abnormal tissue,the cells of which contain a transcription factor that allowsreplication of a replication-conditional vector, or are deficient for aninhibitory factor for transcription.

In this method, a tissue biopsy is, explanted, a replication-conditionalvector is introduced into the cells of the biopsy, and vector DNAreplication in the cells is quantitated. Accordingly, a method isprovided for screening tissue for the presence of factors, that allowvector replication, or for a deficiency of a factor that inhibitstranscription. Such a screen is useful, among other things, foridentifying tissue, prior to treatment, which will be amenable totreatment with a particular vector to be replicated in the tissue.

Therefore, a method is provided for assaying vector utility fortreatment by removing a tissue biopsy from a patient, explanting thebiopsy into tissue culture, introducing the replication-conditionalvector into the biopsy, and assaying vector replication in the cells ofthe biopsy.

Testing or screening of tissues includes an assay for vector nucleicacid replication or for virus replication, when the vector is capable offorming infectious virions.

Thus, the invention provides a method for screening a tumor fortranscription regulatory functions that allow vector replication or forthe absence of these functions which would normally prevent thereplication of a virus vector.

However, any abnormal tissue can be screened for the functions describedabove by an assay for nucleic acid or virus replication.

Producer Cells

In a further embodiment of the invention, a cell is provided whichcontains a virion produced in the cell by replication in the cell of thereplication-conditional vectors of the present invention. Thus, theinvention provides “producer cells” for the efficient and safeproduction of recombinant replication-conditional vectors for furtheruse for targeted gene therapy in vivo.

One of the major problems with the currently available producer cells isthat such cells contain, in the genome, viral sequences that providecomplementing functions for the replicating vector. Because the cellcontains such sequences, homologous recombination can occur between theviral sequence in the genome and the viral vector sequences. Suchrecombination can regenerate recombinant wild-type viruses whichcontaminate the vector or virus preparation produced in the producercell. Such contamination is undesirable, as the wild-type viruses orvectors can then replicate in non-target tissue and thereby impair orkill non-target cells. Therefore, one of the primary advantages of theproducer cells of the present invention is that they do not containendogenous viral sequences homologous to, sequences found in the vectorto be replicated in the cells. The absence of such sequences avoidshomologous recombination and the production of wild-type viralrecombinants that can affect non-target tissue.

Accordingly, the invention embodies methods for constructing andproducing replication-conditional virions in a cell comprisingintroducing the replication-conditional vector of the present inventioninto the cell wherein the genome of the cell is devoid of vectorsequences, replicating the vector in the cell, forming the virion, andpurifying the virion from the cell. Preferred vectors are DNA viralvectors, including but not limited to herpesvirus, papillomavirus,hepatitis virus, and papovavirus vectors. In preferred embodiments ofthe invention, the virion is an adenoviral virion and the vector is anadenoviral vector. In further embodiments of the invention, the cell isa tumor cell.

In a further preferred embodiment, the vector encodes a heterologousgene product such that the virion also encodes the gene product, andwhen the vector or virion are used for gene therapy, the therapy isfacilitated by expression of the heterologous gene product.Alternatively, the producer cell can be used for the production of aheterologous gene product per se encoded by the vector. When the vectorreplicates in the producer cell, the gene product is expressed from themultiple copies of the gene encoding the gene product. Followingexpression, the gene product can be purified from the producer cells byconventional lysis procedures, or secreted from the producer cell byappropriate secretion signals linked to, the heterologous gene by knownmethods. The transduction of cells by adenoviral vectors has beendescribed. Transfection of plasmid DNA into cells by calcium phosphate(Hanahan, D., J. Mol. Biol. 166:577 (1983)), lipofection (Feigner etal., PNAS 84:7413 (1987)), or electroporation (Seed, B., Nature 329:840( )) has been described. DNA, RNA, and virus purification procedures aredescribed (Graham et al., J. Gen. Virol. 36:59-72 (1977).

Preferred hosts for producer cell lines, include but are not limited toHuH7, SW480, BIGF10, HepG2, MCF-7, and SK-MEL2. Primary tumors fromwhich cell lines can be derived, or existing cell lines, can be testedfor the ability to allow replication by means of the tissue-specifictranscriptional regulatory sequence. Any primary tumor could beexplanted and developed into producer cells for the vectors of thepresent invention. As long as the cell does not contain endogenousvector or viral sequences that could recombine with the vector or virusto produce wild-type vector or virus, the cell is potentially useful asa host. It is understood that any cell is potentially useful, not onlytumor cells.

The ultimate goal for a producer cell line, and particularly anadenoviral producer line, is to produce the highest yield of vector withthe least possibility of contamination by wild-type vector. Yielddepends upon the number of cells infected. Thus, the more cells that itis possible to grow and infect, the more virus it is possible togenerate. Accordingly, candidate cells would have a high growth rate andwill grow to a high density. The cell should also have a high amount ofviral receptor so that the virus can easily infect the cell. Anothercharacteristic is the quality of the vector produced (i.e., thepreparation should not include a high amount of non-infectious viralparticles). Accordingly, candidate producer cells would have a lowparticle-to-plaque-forming-unit ratio. Thus, these cells are a preferredcell type for deriving a producer cell line. Primary explants or theknown cell lines can be used.

Thus, such obtainable cells can serve as producer cells for recombinantreplication-conditional vectors, viruses, and gene products.

Introduction of Vectors into Cells

A variety of ways have been developed to introduce vectors into, cellsin culture, and into cells and tissues of an animal or a human patient.Methods for introducing vectors into mammalian and other animal cellsinclude calcium phosphate transfection, the DEAE-dextran technique,microinjection, liposome mediated techniques, cationic lipid-basedtechniques, transfection using polybrene, protoplast fusion techniques,electroporation and others. These techniques are well known to those ofskill, are described in many readily available publications and havebeen extensively reviewed. Some of the techniques are reviewed inTranscription and Translation, A Practical Approach, Hames, B. D. andHiggins, S. J., eds., IRL Press, Oxford (1984), herein incorporated byreference in its entirety, and Molecular Cloning, Second Edition,Maniatis et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), herein incorporated by reference in its entirety.

Several of these techniques have been used to introduce vectors intotissues and cells, in animals and human patients. Chief among these havebeen systemic administration and direct injection into sites in situ.Depending on the route of administration and the vector, the techniqueshave been used to introduce naked DNA, DNA complexed with cationiclipid, viral vectors and vector producer cell lines into normal andabnormal cells and tissues, generally by direct injection into atargeted site.

The aforementioned techniques for introducing polynucleotide, viral andother vectors into cells in culture, in animals and in patients can beused to develop, test and produce, as well as use vectors in accordancewith the invention. For instance, cells containing a vector introducedby these methods can be used for producing the vector. In addition,cells containing a vector can be used as producer-cells and introducedinto cells or tissues of an animal to produce the vector in situ.

Assay of DNA and Viral Replication

Replication of a polynucleotide, viral or other vector can be assayed bywell-known techniques. Assays for replication of a vector in a cellgenerally involve detecting a polynucleotide, virions or infectivevirus. A variety of well-known methods that can be used for thispurpose, involve determining the amount of a labelled substrateincorporated into a polynucleotide during a given period in a cell.

When replication involves a DNA polynucleotide, ³H-thymidine often is,used as the labelled substrate. In this case, the amount of replicationis determined by separating DNA of the vector from the bulk of cellularDNA and measuring the amount of tritium incorporate specifically intovector DNA.

Replication of a polynucleotide vector also may be detected by lysing orpermeating cells to release the polynucleotide, then isolating thepolynucleotide and quantitating directly the DNA or RNA that isrecovered. Polynucleotide replication also may be detected byquantitative PCR using primers that are specific for the assaypolynucleotide.

Virions may be assayed by EM counting techniques well known to the art,by isolating the virions and determining protein and nucleic acidcontent, and by labelling viral genomic polynucleotides or virionproteins and determining the amount of virion from the amount ofpolynucleotide or protein.

It is well known that virions may not all be viable and whereinfectivity is important, infectious titer may be determined bycytopathic effect or plaque assay.

Any of these well-known techniques, among others, can be employed toassay replication of a vector in a cell or tissue in accordance with theinvention. It will be appreciated that different techniques will bebetter suited to some vectors than others and to some cells or tissuesthan others.

Having thus described herein the invention in general terms, thefollowing examples are presented to illustrate the invention. Examples1-4 show the replacement of the constitutive E1A promoter on anadenoviral vector with a tumor-specific promoter. Constructs made thisway have the E1a protein expressed only in tumor cells and therefore,will replicate only in tumor cells.

Example 1 The Hepatoma-Specific Promoter, α-Fetoprotein Promoter, Linkedto E1a

The α-fetoprotein promoter has been previously shown to be highly activein hepatoma cells and silent in adult hepatocytes and other adulttissues. A 4.9 kb α-fetoprotein promoter containing construct was usedto derive the promoter. Alternatively, the promoter could also be madebased on available references.

The adenovirus shuttle plasmid pAVS21.TK1 (FIG. 1), which has the TKgene under the control of the 4.9 kb α-fetoprotein promoter, was madeexactly as described in FIGS. 11 and 12 of U.S. patent application Ser.No. 08/444,284 of Chiang et al for “Gene Therapy of HepatocellularCarcinoma Through Cancer-Specific Gene Expression”, filed on May 18,1995, which is incorporated herein by reference for its relevantteaching. pAVE1a02i (FIG. 1) which places the E1a/E1b genes under thecontrol of the α-fetoprotein promoter in an adenovirus shuttle plasmidwas cloned by purifying a restriction fragment which contained the E1acoding region only and all of E1b gene by cleaving the plasmid pSE280-E1(FIG. 1) with SpeI and MunI and ligating this to pAVS21.TK1 cleaved withMunI and NheI. Plasmid SE280-E1, which contains the E1A ORF and all ofE1b, was constructed as described in U.S. patent application Ser. No.08/458,403 to Kadan et al. for “Improved Adenoviral Vectors and ProducerCells,” filed Jun. 2, 1995, which is incorporated herein by referencefor its relevant teaching. pAVE1a02i is cotransfected with the largeClaI fragment of Add1327 by standard methods into 293 cells to generaterecombinant virus.

Construction of a Virus with the Hepatoma-Specific AFP Promoter OperablyLinked to the E1a Gene

The adenovirus AVE1a04i was constructed by homologous recombination ofthe shuttle plasmid, pAVE1a04i (See FIG. 2), with the large (Cla1)fragment of AV1lacZ4 DNA in 293 cells. The construction of the plasmidpAVE1a02i is described above. The construction of pAVE1a04i is almostidentical to that of pAVE1a02i. pAVE1a02i contains the entire AFPpromoter. pAVE1a04i utilizes a derivative of this promoter, which hassix silencer elements and a duplicated enhancer region.

The plasmid pAF(AB)₂(S_(d))₆—CAT was constructed by placing six copiesof the distal silencer immediately upstream of the basal 200 base pairAFP promoter. Two copies of the enhancer AB region, in oppositeorientation, are placed immediately upstream of the silencer elements.This promoter, extending from the enhancer element through the basal AFPpromoter, was used to make the AV/AFP short E1a virus with the shuttleplasmid described herein. The distal silencer element, the basalpromoter, and the enhancer elements are as described in Nakabayashi etal. (Molec. & Cell. Biol. 11:5885-5893 (1991)).

The plasmid pAVE1a04i was grown in STBL2 cells and was purified bystandard cesium banding methods prior to use in transfection. GenomicAV1lacZ4 DNA was isolated from cesium gradient-purified virus (hereindescribed). The AV1lacZ4 purified virus was digested with proteinase Kand the DNA isolated by phenol/chloroform extraction. The purified DNAwas digested with Cla1 and the large fragment was isolated by gelelectrophoresis and quantified. 5 μg of the plasmid pAVE1a04i and 2.5 μgof the large ClA1 fragment of AV1lacZ4 were co-transfected into 293cells using a calcium phosphate-mediated transfection procedure(Promega, E1200 kit). The transfection plate was overlayered with a 1%agarose overlay and incubated until plaques formed. Once plaques hadformed, they were picked and the virus was released into 500 μl of IMmedia by alternate cycles of freezing and thawing (5×). The eluted viralplaques were reamplified on A30 cells for 48 hours and then the cellswere lysed for use in screening by PCR.

Primers specific for the short AFP (sAFP) promoter in plasmid pAVE1a04iwere used to identify the putative plaques. FIG. 2A shows that viralplaques contain a sAFP-specific band of the predicted molecular weightand specific for the sAFP primers. To confirm that this recombinantvirus was not contaminated with Ad5d1327 (wild type), E1a primers wereused. FIG. 2B demonstrates that no wild type virus was present and thatpAVE1a04i plasmid sequences were present in the recombinant virus. FIG.2C demonstrates that little or no AV1lacZ4 was present. The dataindicate the construction of a virus with E1a under control of atissue-specific promoter and that the virus is capable of replication inA30 cells.

Individual plaques were grown in A30 cells and analyzed by PCR for thepresence of the AFP promoter (FIG. 2). The arrow indicates theAFP-specific band generated from PCR. The figure shows that the band ispresent in each of the viruses in the selected plaques (L6, L10, L11, M1and M2). The control in the experiment was an A30 cell lysate, expectednot to contain the band. The experiment also included the PCR reactionwith the plasmid pAVE1a04i (the shuttle plasmid from which the virus wasmade and which therefore should produce the AFP-specific fragment).Thus, FIG. 2A confirms the presence of a recombinant virus containingthe AFP promoter. FIGS. 2B and 2C confirm that these results were notthe result of contamination in the individual plaques. FIG. 2B usesE1a-specific primers to detect the presence of any contaminatingwild-type virus. The arrow shows the band produced with E1a-specificprimers. The figure shows that none of the recombinant viruses producedthe relevant band. FIG. 2C confirms that there is no AV1.lacZcontamination in the viral plaques (since the viruses were made usingAV1.lacZ DNA). The figure indicates that only the lane containingAV1.lacZ DNA produced the band.

Tissue-Specific Viral Replication

Cytopathic viral lysate of this virus (“AVAFPE1a”) was serially dilutedin logs of 10 on A549.30 cells, A549 cells, and HuH 7 cells. A549.30cells express the E1a from the glucocorticoid receptor element (GRE)promoter in the presence of dexamethasone since this construct isintegrated into the genome of this cell line. Thus, any E1a-deletedvirus or any virus not expressing E1a should be able to replicate inthis cell line. This has previously been shown for E1-deleted vectors,(unpublished communication). As can be seen from FIGS. 3A and 3D, theAVAFPE1a vector replicates in the infected cells as indicated bycharacteristic cytopathic effects and spreading of cell death. The A549cells do not express. AFP and should not be capable of transactivatingthe AFP promoter. In addition, A549 cells do not express E1a Thus,AVAFPE1a should not be able to replicate in this cell line. As can beseen from FIGS. 3B and 3E, both uninfected and infected wells appearidentical with no characteristic cytopathic effects or spreadingobserved at all dilutions tested. HuH 7 cells do express AFP, shouldtransactivate the AFP promoter, and should make E1a with subsequentreplication. As shown in FIGS. 3C and 3F, AVAFPE1a clearly replicates,as indicated by the cytopathic effects. In addition, on several wells ofinfected HuH 7 cells, the replication began with a single plaque whichspread throughout the rest of the well within one week. All HuH 7 wellsshowing cytopathic effects were tested by PCR and demonstrated to befree of wild-type virus and AV1LacZ4 virus, and to contain an intact AFPpromoter. These data clearly indicate that a virus has been constructedthat is capable of replicating specifically in tumor cells expressingAFP.

Example 2 The Breast Cancer-Specific DF3-Mucin Enhancer

The DF3 breast carcinoma associated antigen (MUC1) is highlyoverexpressed in human breast carcinomas. The expression of the gene isregulated at the transcriptional level. The DNA sequence between−485-588 is necessary and sufficient for conferring a greater than10-fold increase in transcription of the reporter gene CAT when placedimmediately upstream of a basal promoter derived from the Herpesvirus TKpromoter in transient transfection assays performed in the human breastcancer cell line MCF-7. A specific, transcription factor which binds tothis region of DNA has also been found within cells derived from thebreast cancer cell line MCF-7 but not a non-breast cancer cell lineHL-60. The same region of DNA has been found to promote breastcancer-specific expression of the TK gene in the context of a retroviralconstruct or an adenoviral construct.

The DF3 enhancer from −598 to −485 (obtained from GenBank) wassynthesized by constructing four oligonucleotides synthesized in such away as they would overlap and anneal. The oligonucleotides are shown inTable 1. Additional restriction sites were added on both ends for futureease of cloning. One end was kept blunt to enable cloning into the SmaIsite of the vector pTK-Luc. This vector contains the basal promoter ofthe Herpesvirus TK gene which gives low level basal activity in avariety of cells. It was used as a source of this basal promoter. Theother end had an overlapping BglII site for ease in cloning into theBglII site of pTK-Luc. 1,000 ng of each oligonucleotide were annealed in0.017 M Tris, pH 8.0, 0.16 M NaCl in a total volume of 26.5 μl byheating at 95° C. for two minutes and allowing to cool to roomtemperature after several hours. Finally, 1 μl of this mixture wasligated to 100 ng of previously SmaI/BglII and glass milk (BIO 101)purified vector by standard conditions. Following transformation intoDH5α cells (GIBCO), colonies were screened for the presence of theinsert by standard restriction digests. DNA derived from this vector isthen cleaved with HindIII and blunted by Klenow. It is then cleaved byAscI. This fragment, which contains the DF3 enhancer lined to the basalTK promoter, is then purified by agarose gel electrophoresis and glassmilk and ligated to the plasmid pAVE1a02i, cleaved with Spe I andblunt-ended with AscI and purified as above. The resultant plasmid hasthe E1A gene product under the control of the DF3 enhancer and basal TKpromoter and is in an adenoviral shuttle plasmid. 5 μg of this plasmid,pAVE1a03i, is cotransfected with 5 μg of the right ClaI fragment arm,derived from Add1327, into 293 cells. Plaques are screened for theexpected recombinant virus by standard methods.

A crude virus lysate is used to infect MCF-7 at an MOI of 10. Virusstocks are confirmed to replicate specifically in breast cancer cells bystandard methods. Virus is scaled up in MCF-7 cells and/or 293 cells asdescribed for scaleup and purification on 293 cells. Virus stocks aretested for replication in vivo by using a mode mouse model of MCF-7 and,as a negative control, a cervical cancer (Hela) derived tumor is used.Virus is tested for a recombinational event in 293 cells which wouldgenerate a wild-type virus by PCR assay of the original E1A promoterwhich would only be in a wild-type virus. A variety of other human andrat breast cancer cell lines and non-related cell lines are also tested.The TK gene can be inserted into the E3 region and have TK driven eitherby the E1A-dependent promoter present there or under the control of theRSV or CMV promoter.

Example 3 The Melanoma-Specific Tyrosinase Promoter

PCR primers and PCR were used to clone a fragment of DNA 800 bp upstreamof the tyrosinase gene from mouse genomic DNA using PFU and thedescribed primers as described by Stratogene. The resultant PCR fragmentwas cloned into pCRSCRIPT and then recloned into pAVE1a02i by digestingthe new plasmid with AscI/SpeI and pAVE1a01i with AscI/SpeI and ligatingthe two together. The final shuttle plasmid, pAVE1a04i, which has,E1a/E1b under the control of the tyrosinase promoter, is utilized tomake a recombinant virus identically as described above.

Example 4 The Colon Cancer-Specific CEA Promoter

The CEA promoter was cloned from human genomic DNA as described aboveand cloned in a similar way into the pAVE1a01i plasmid using the primersshown in Table 1. The final shuttle plasmid, pAVE1a05i, is used togenerate recombinant virus as described above.

Example 5 A. Replacing the Promoter of E2a on an Adenoviral Vector witha Tumor Specific Promoter

Constructs made as above will have the E2a protein (essential for viralreplication expressed only in tumor cells. Therefore, replication of thevector occurs only in tumor cells. All four of these very specificpromoters (in the examples above) are used to place the E2a codingregion obtained from pSE280-E2a (see U.S. patent application Ser. No.08/458,403 of Kadan et al., “Improved adenoviral vectors and producercells” filed Jun. 2, 1995) under the control of that tumor-specificpromoter. The resultant plasmid is recombined with Add1327, usingstandard methods of homologous recombination. The final virus is grownin the cell lines described in the aforementioned patent application orin the tumor specific cell lines. The E2a protein, because it is neededin stoichiometric amounts, has the ability to regulate the degree ofreplication over a broad range. This is desirable for therapy. Themethods used are the same as those described for E1a. The difference isthat a shuttle plasmid is used that places E2a under the control of thetumor specific promoter and returns it to a virus backbone (byhomologous recombination) that has the E2a and E3 genes deleted.

B. Replacement of Other Therapeutic Toxic Genes into the Tumor-SpecificReplication Competent Vectors

Genes such as TK, cytokines, or any therapeutic genes can be placed intothe E3 region of the vector backbone by standard plasmid constructionand homologous recombination. Those genes can be placed under thecontrol of an E1a-dependent promoter, or a constitutive promoter such asRSV or CMV.

The disclosures of all patents, publications (including published patentapplications), and database accession numbers referred to in thisspecification are specifically incorporated herein by reference in theirentirety to the same extent as if each such individual patent,publication, and database accession numbers were specifically andindividually indicated to be incorporated by reference in its entirety.TABLE 1 Oligonucleotide Primers for Constructing Tissue- SpecificPromoters 1. DF3 (Breast Cancer) (SEQ ID NO: 1) 5′ GGG CGC GCC CTG GAAAGT CCG GCT GGG GCG GGG ACT GTG GGT TTC AGG GTA GAA CTG CGT GTG GAA 3′(SEQ ID NO: 2) 5′ CGG GAC AGG GAG CGG TTA GAA GGG TGG GGC TAT TCC GGGAAG TGG TGG GGG GAG GGA ACT AGT A 3′ (SEQ ID NO: 3) 5′ GAT CTA CTA GTTCCC TCC CCC CAC CAC TTC CCG GAA TAG CCC CAC CCT TCT AAC CGC TCC CTG 3′(SEQ ID NO: 4) 5′ TCC CGT TCC ACA CGC AGT TCT ACC CTG AAA CCC ACA GTCCCC GCC CCA GCC GGA CTT TCC AGG GCG CGC CC 3′ 2. Tyrosinase (Melanoma)(SEQ ID NO: 5) 5′ GAC CCG GGC GCG CCG GAG CAG TGC TAT TCA AAC CAT CCA G3′ (SEQ ID NO: 6) 5′ CGA GAT CTA CTA GTT CTG CAC CAA TAG GTT AAT GAG TGTC 3′ 3. CEA Promoter (Hepatocellular Carcinoma) (SEQ ID NO: 7) 5′ GACCCG GGC GCG CCT CTG TCA CCT TCC TGT TGG 3′ (SEQ ID NO: 8) 5′ CGA GAT CTACTA GTT CTC TGC TGT CTG CTC TGT C 3′

1. A vector capable of tissue-specific replication comprising: a tissue-specific transcriptional regulatory sequence operably linked to the coding region of a gene that is essential for replication of said vector. 2-40. (canceled) 